Understanding termite digestion could help biofuels, insect control

February 18, 2013

WEST LAFAYETTE, Ind. – A
termite's own biology with help from microorganisms called protists, are keys
to the insect's digestion of woody material, according to a Purdue University
scientist.

Michael Scharf, the O. Wayne
Rollins/Orkin Endowed Chair in Urban Entomology, studies termite digestion to
improve biofuels production and find better ways to control termites. The U.S.
Department of Agriculture estimates the cost of controlling termites and
repairing damaged homes is $2 billion each year in the United States.

Much of the study on how
termites break down woody materials, which focused on the symbiotic
relationship between the insect and the bacteria living in its gut, found that
bacteria apparently have little, if anything, to do with termite digestion.

Scharf and collaborators at
the University of Florida wanted to see how diet affected those bacteria. If
the bacteria play a role in digestion, the type of materials the insect eats
should affect the composition of the bacterial community living in the termite
gut.

More than 4,500 different
species of bacteria were cataloged in termite guts. When multiple colonies of
termites were independently fed diets of wood or paper, however, those bacteria
were unaffected.

"You would think diet
would cause huge ecological shifts in bacterial communities, but it didn't. We
didn't detect any statistical differences," Scharf said.

What they did see were far
more significant changes in gene expression in the termites and the protists
that live in the insects' guts along with the bacteria.

"The bacteria
communities seem very stable, but the host and the protozoa gene expression are
changing a lot based on diet," Scharf said.

The scientists looked at
10,000 gene sequences from the termites and protists to determine which genes
were expressed based on differing diets. Termites and protists fed woody and
lignin-rich diets changed expression of about 500 genes, leading Scharf to believe
those genes might be important for breaking down lignin, a rigid material in
plant cell walls that isn't easily broken down when making biofuels.

"We see much more of
the playing field now," Scharf said.

Understanding which genes
are involved in digestion should help researchers track down the enzymes that
actually break down woody materials in termite digestion. Those enzymes may be
tools scientists could use to better break down biomass and extract sugars
during biofuel production.

The National Science
Foundation, the Consortium for Plant Biotechnology Inc. and the U.S. Department
of Energy funded the research.

The findings were detailed
in three papers published in the journals Molecular
Ecology, Insect Molecular Biology,
and Insect Biochemistry and Molecular
Biology.

Reticulitermes flavipes (Isoptera:
Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant
lignocellulosic diets through nutritional symbioses with gut-dwelling
prokaryotes and eukaryotes. In the R.
flavipes hindgut, there are up to 12 eukaryotic protozoan
symbionts; the number of prokaryotic symbionts has been estimated in the
hundreds. Despite its biological relevance, this diverse community, to date,
has been investigated only by culture- and cloning-dependent methods. Moreover,
it is unclear how termite gut microbiomes respond to diet changes and what
roles they play in lignocellulose digestion. This study utilized
high-throughput 454 pyrosequencing of 16S V5-V6 amplicons to sample the hindgut
lumen prokaryotic microbiota of R. flavipes
and to examine compositional changes in response to lignin-rich and lignin-poor
cellulose diets after a 7-day feeding period. Of the ~475,000 high-quality
reads that were obtained, 99.9% were annotated as bacteria and 0.11% as
archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia
(19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%),
Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and
Tenericutes (1.3%). The R. flavipes
hindgut lumen prokaryotic microbiota was found to contain over 4,761
species-level phylotypes. However, diet-dependent shifts were not statistically
significant or uniform across colonies, suggesting significant environmental
and/or host genetic impacts on colony-level microbiome composition. These
results provide insights into termite gut microbiome diversity and suggest that
(i) the prokaryotic gut microbiota is much more complex than previously
estimated, and (ii) environment, founding reproductive pair effects and/or host
genetics influence microbiome composition.

ABSTRACT

Comparative Metatranscriptomic Signatures of
Wood and Paper Feeding in the Gut of the Termite Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Termites are highly
eusocial insects that thrive on recalcitrant materials like wood and soil and
thus play important roles in global carbon recycling and also in damaging
wooden structures. Termites, such as Reticulitermes
flavipes (Rhinotermitidae), owe their success to their ability to
extract nutrients from lignocellulose (a major component of wood) with the help
of gut-dwelling symbionts. With the aim to gain new insights into this
enzymatic process we provided R. flavipes with a complex lignocellulose
(wood) or pure cellulose (paper) diet and followed the resulting differential
gene expression on a custom oligonucleotide-microarray platform. We identified
a set of expressed sequence tags (ESTs) with differential abundance between the
two diet treatments and demonstrated the source (host/symbiont) of these genes,
providing novel information on termite nutritional symbiosis. Our results
reveal: (1) the majority of responsive wood- and paper-abundant ESTs are from
host and symbionts, respectively; (2) distinct pathways are associated with
lignocellulose and cellulose feeding in both host and symbionts; and (3) sets
of diet-responsive ESTs encode putative digestive and wood-related
detoxification enzymes. Thus, this study illuminates the dynamics of termite
nutritional symbiosis and reveals a pool of genes as potential targets for
termite control and functional studies of termite-symbiont interactions.

Lignin is a component
of plant biomass that presents a significant obstacle to biofuel production. It
is composed of a highly stable phenylpropanoid matrix that, upon degradation,
releases toxic metabolites. Termites have specialized digestive systems that
overcome the lignin barrier in wood lignocellulose to efficiently release
fermentable simple sugars; however, how termites specifically degrade lignin
and tolerate its toxic byproducts remains unknown. Here, using the termite Reticulitermes flavipes and its symbiotic
(protozoan) gut fauna as a model system, we used high throughput Roche
454-titanium pyrosequencing and proteomics approaches to (i) experimentally
compare the effects of diets containing varying degrees of lignin complexity on
host-symbiont digestome composition, (ii) deeply sample host and symbiont
lignocellulase diversity, and (iii) identify promising lignocellulase
candidates for functional characterization. In addition to revealing over 9,500
differentially expressed transcripts related to a wide range of physiological
processes, our findings reveal two detoxification enzyme families not generally
considered in connection with lignocellulose digestion: aldo-keto reductases and catalases. Recombinant versions of two
host enzymes from these enzyme families, which apparently play no roles in
cellulose or hemicellulose digestion, significantly enhance lignocellulose
saccharification by cocktails of host and symbiont cellulases. These hypothesis-driven
results provide important new insights into (i) dietary lignin as a xenobiotic
challenge, (ii) the complex mechanisms used by termites to cope with their
lignin-rich diets, and (iii) novel lignin-targeted enzymatic approaches to
enhance biofuel and biomaterial production.